Sequencing

So far, most DNA sequencing has been performed using the chain termination method developed by Frederick Sanger.

This technique uses sequence-specific termination of a DNA synthesis reaction using modified nucleotide substrates.

Determining the sequence is therefore useful in fundamental research into why and how organisms live, as well as in applied subjects.

[3] Carlson accurately predicted the doubling time of DNA sequencing technologies (measured by cost and performance) would be at least as fast as Moore's law.

[4] Carlson curves illustrate the rapid (in some cases hyperexponential) decreases in cost, and increases in performance, of a variety of technologies, including DNA sequencing, DNA synthesis, and a range of physical and computational tools used in protein expression and in determining protein structures.

In this sequencer four different vessels are employed, each containing only of the four dideoxyribonucleotides; the incorporation of the chain terminating nucleotides by the DNA polymerase in a random position results in a series of related DNA fragments, of different sizes, that terminate with a given dideoxiribonucleotide.

The fragments are then size-separated by electrophoresis in a slab polyacrylamide gel, or more commonly now, in a narrow glass tube (capillary) filled with a viscous polymer.

This problem has been significantly reduced with the introduction of new enzymes and dyes that minimize incorporation variability.

This is changing rapidly due to the increasing cost-effectiveness of second- and third-generation systems from Illumina, 454, ABI, Helicos, and Dover.

In the array-based method (commercialized by 454 Life Sciences), single-stranded DNA is annealed to beads and amplified via EmPCR.

These DNA-bound beads are then placed into wells on a fiber-optic chip along with enzymes which produce light in the presence of ATP.

Addition of one (or more) nucleotide(s) results in a reaction that generates a light signal that is recorded by the CCD camera in the instrument.

This fraction can be removed in vitro, however, to enrich for the messenger RNA, also included, that usually is of interest.

Determining part of a protein's amino-acid sequence (often one end) by one of the above methods may be sufficient to identify a clone carrying this gene.

Methods for the structure determination of oligosaccharides and polysaccharides include NMR spectroscopy and methylation analysis.